Gsno reductase inhibitor as an adjunct therapy with remote ischemic conditioning and thrombolytic reperfusion therapies in stroke

ABSTRACT

Stroke patients with comorbidities, such as diabetes, hypertension, or other conditions associated with cardiovascular, pulmonary or renal disease do not respond well to current treatments for stroke. An inhibitor of S-nitrosoglutathione reductase (GSNOR) is administered as an adjunct therapy with remote ischemic conditioning (RIC) to treat a stroke in a subject having at least one comorbidity, such as diabetes and hypertension. When performed in conjunction with GRI therapy and RIC, intravenous tissue plasminogen activator therapy (IVT) for thrombolysis and/or endovascular thrombectomy (EVT) for clot retrieval are effective in subjects with comorbidities, even after five hours post-onset of stroke.

BACKGROUND OF THE INVENTION Field of the Invention

The invention generally relates to a treatment for stroke when remoteischemic conditioning is not effective. The invention further relates toa method for repurposing an inhibitor of S-nitrosoglutathione reductaseas an adjunct therapy to delayed or late IVT (beyond 3 hours) in stroke,particularly for ischemic stroke with comorbidities.

Background

Acute ischemic stroke (AIS), often due to a thromboembolic (TE)occlusion in cerebral arteries, results in reduced cerebral blood flow(CBF), brain tissue hypoxia/ischemia and subsequent progression ofcerebral infarction and brain cell death. To date, the FDA has approvedonly two reperfusion therapies in AIS: intravenous tissue plasminogenactivator therapy (IVT) for thrombolysis within 3 hours of stroke andendovascular thrombectomy (EVT) for mechanical clot retrieval. These twotherapies re-open large occluded blood vessels and reinstate CBF inthem. However, neither IVT nor EVT guarantee the restoration ofmicrocirculatory reflow, which is critically needed to eventuallyreoxygenate the ischemic region. Thus, despite a successful reperfusiontherapy in stroke, brain tissue hypoxia may persist, which remains acritical barrier in improving post-stroke outcomes. Therefore, ischemicstroke remains a significant cause of adult mortality and a leadingcause of adult disability worldwide. The FDA and NIH/NINDS strokeresearch progress related committees strongly recommend the developmentand repurposing of existing therapies to treat stroke (Fisher et al.Stroke 2009, 40(6):2244-2250). It is also recommended that the newpromising adjunct therapy should be effective alone to reduceischemia-reperfusion (IR) injury, should be safe to utilize as a fieldtherapy without requiring any state-of-art facilities such as in remotecommunity hospitals for primary care, during transport in ambulance,Emergency Department and any acute care situation. Most importantly, anew and/or repurposed therapy also should not contradict and prevent theuse of IVT/EVT.

Nitric oxide (NO) is a freely diffusible endogenous small molecule whichis produced by the NO-synthase (NOS) enzymes. Endothelial NOS (eNOS)remains the major producer of vascular/endothelial NO which relaxesvascular smooth muscles cells and improves tissue oxygenation.Endogenous NO can be present as nitrate (NO3⁻)/nitrite (NO2⁻) orpreserved as s-nitrosylated proteins (—SH to —SNO) such as glutathioneGSH>GSNO and hemoglobin Hb>HbSNO. Thus, GSNO and HbSNO were recognizedas the major chariots and sources of bioactive endogenous NO in the formof —SNO adducts. Exogenous NO-inhalation, which increases levels ofGSNO/HbSNO, was one of the earliest therapies incidentally identified totreat angina and later tested in ischemic stroke (Lundberg et al. NatRev Drug Discov. 2008; 7(2):156-167). However, NO-inhalation therapyincreases risk of blood pressure lowering which can cause hemorrhagictransformation (HT) to worsen stroke outcomes. On the other hand, NO inthe form of conventional NO3⁻/NO2⁻ therapy recently showed no benefitsin stroke clinical trial (Bath et al. Cochrane Database Syst Rev. 2017;4:CD000398).

Others and we recently reported that remote ischemic conditioning (RIC)modulates endogenous NO-metabolome, enhances CBF and improves outcomesafter stroke/ischemic brain injuries (Hess et al. Acta Neurochir Suppl.2016; 121:45-48; Hoda et al. Stroke. 2012; 43(10):2794-2799; Hoda et al.Transl Stroke Res. 2014, 5(4):484-90). However, it remains a concernthat in a larger population of stroke patients often accompanied withmost common comorbidities (such as hypertension and diabetes), RIC mayfail to protect. Indeed, it is evident from a latest preliminary smallclinical trial report that RIC therapy remained neutral in effect instroke patients, likely indicating that the “no-effects” outcome incomorbid stroke patients might have neutralized or skewed the overalloutcome of the trial (Pico et al. Int J Stroke. 2016; 11(8):938-943).

Thus, there is a need for an adjuvant treatment that can enhancebenefits of the current FDA-approved reperfusion therapies to furtherimprove functional outcomes after stroke. Compounding this lack oftreatment options, most exogenous therapies have failed in stroke trialsin the past four decades aside from IVT/EVT, so the problem ofmicrocirculatory no-reflow persists. In particular, there is a need foran effective treatment for patients with comorbidities to improverecovery from stroke and reduce ischemic brain injury by promotingreperfusion and restoring microcirculatory flow because comorbiditiessuch as diabetes alters the vascular patency and increases the risk ofHT during ischemic stroke, particularly in response to late IVT.

SUMMARY OF THE INVENTION

The invention is a method of treatment for a stroke and is particularlysuited for treating a subject who also has at least one comorbidcondition, such as diabetes and/or hypertension. The treatment comprisesadministering a therapeutically effective amount of anS-nitrosoglutathione reductase (GSNOR) inhibitor, wherein thetherapeutically effective amount is sufficient to inhibit brain tissueinjury. The GSNOR inhibitor is an adjunct therapy to be administered inconjunction with the RIC therapy, particularly in stroke withcomorbidities such as stroke patients accompanied with diabetes and/orhypertension. GSNOR inhibitors that may be used include N6022,cavosonstat, N91115, N6338, as well as any other GSNOR inhibitor. TheGSNOR inhibitor may be administered prior to performing RIC therapy orconcurrently with the RIC therapy. The RIC therapy is performedaccording to an FDA-approved protocol and/or by using an FDA-approveddevice.

In one embodiment, the method further comprises the step ofadministering a therapeutically effective amount of IVT, wherein thetherapeutically effective amount is sufficient to achieve thrombolysis,and/or administering EVT for clot retrieval. The combination of GRItherapy and RIC therapy with EVT and/or IVT is effective even when the“window of opportunity” of 3 hours has passed since the time of onset ofstroke, which is when the risk of HT would otherwise will increase dueto late IVT.

In another embodiment, the subject having comorbid conditions issuspected to be suffering from a stroke or is at the risk of sufferingan imminent stroke with comorbidities and lacking FDA-approvedreperfusion interventions (IVT/EVT) for at least 3 h. Stroke in subjectshaving comorbidities is particularly dangerous and more likely to resultin exacerbated brain injury and HT than subjects not havingcomorbidities. In particular, the patient is suffering from at least onecomorbid condition, such as diabetes, type II diabetes, hypertension,cardiac disease, coronary artery disease, arteriosclerosis,atherosclerosis, myocardial infarct, congestive heart failure,peripheral vascular disease, dementia, cerebrovascular disease, chronicpulmonary disease, congestive obstructive pulmonary disease, kidneydisease, kidney failure, chronic liver disease and metabolic syndrome.In one embodiment, the subject is suffering from the comorbid conditionsof diabetes and hypertension, and the GSNOR inhibitor is administered asan adjunct therapy to RIC therapy to enhance the efficacy and benefitsof RIC. Optionally, IVT and/or EVT are also administered to dissolveand/or remove a thrombus or blood clot from an artery, thereby restoringperfusion of the brain region in which blood flow was being blocked bythe thrombus/clot.

As a result of the treatment, ischemic injury and brain tissue infarctcan be reduced, minimized or avoided, and functional activity that wouldotherwise be impaired is restored, at least in part. Functionalactivities in humans include cognitive, speech and motor skills that areneeded for normal everyday living and contribute to quality of life.

In one embodiment, the invention is a method of restoring brainmicrocirculatory flow in a subject suffering from at least one comorbidcondition, comprising the step of administering in combination of an RICtherapy and a therapeutically effective amount of an GSNOR inhibitor,wherein the therapeutically effective amount is sufficient to allowreperfusion of a brain region wherein the brain microcirculatory flowwas previously blocked. The GSNOR inhibitor may be administered prior toinitiation of the RIC therapy, or the GSNOR inhibitor may beadministered concurrently with delivery of the RIC therapy.

In another embodiment, the invention is a method of restoring brainmicrocirculation in a subject suffering from at least one comorbidcondition, comprising the step of administering in combination RICtherapy and a therapeutically effective amount of an GSNOR inhibitor,wherein the therapeutically effective amount is sufficient to allowreperfusion of a brain region wherein the brain microcirculatory flowwas previously blocked. Optionally, the RIC and GRI therapy may befollowed by administration of IVT for thrombolysis and/or EVT for clotretrieval.

Other features and advantages of the present invention will be set forthin the description of invention that follows, and in part, will beapparent from the description or may be learned by practice of theinvention. The invention will be realized and attained by thecompositions and methods particularly pointed out in the writtendescription and claims hereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate embodiments of the invention and,together with a general description of the invention given above, andthe detailed description given below, serve to explain the invention.

FIG. 1A-1D shows the effect of embolic middle cerebral artery (MCA)occlusion (eMCAo) on GSNOR protein expression and activity. 1A showssemi-quantitative densitometry of an immunoblot for GSNOR proteinnormalized to β-actin protein in whole brain tissue for sham-operatedgroup. 1B shows a representative immunoblot for GSNOR expression inwhole brain tissue. 1C shows an activity assay based on the rate ofGSNO-dependent consumption of NADH in brain. 1D shows an activity assayof rate of GSNO-dependent consumption of NADH in plasma.

FIGS. 2A-2D show the effect of GRI therapy in aged male mice followingphotothrombotic (PT) stroke, a thrombotic model of moderate injury. 2Ashows a representative image of whole brain collected immediately afterstroke to confirm the site of occlusion in MCA region. 2B shows aZ-projection of T2-weighted magnetic resonance image 48 h after stroke.Site of thrombotic occlusion and stroke injury in 2A and 2B,respectively, are indicated with arrow. 2C shows densitometry of coronalsections from brains of treated mice, and 2D shows representativeTTC-stained set of coronal sections (2-μm thick) obtained from the wholebrain and stained freshly.

FIGS. 3A-3D show that a sex difference was found in the basal level ofplasma GSNOR activity. 3A shows GSNOR activity in males and females. 3Bshows plasma GSNOR activity in aged females after PT-stroke. 3C showsdensitometry of infarct volume measured in coronal sections, and 3Dshows a representative TTC-stained set of coronal sections (2 mm thick)obtained from the whole brain and stained freshly.

FIGS. 4A-4D show analyses of mice in a stroke model of permanent MCAocclusion (pMCAo). 4A shows a representative grey scale laser specklecontrast image (LSCI) immediately after stroke. A narrow beam walk testwas used to determine any deterioration of motor function for walkingtime to traverse through the beam length, shown in 4B, and number offoot slips, shown in 4C. 4D shows % CBF for males andovariectomizedfemales (OVX-Fem) treated with vehicle (Veh) or GRItherapy.

FIGS. 5A-5B show representative LSCI images from mice treated withvehicle or GRI therapy. Males are represented in 5A and OVX-female miceare represented in 5B.

FIGS. 6A-6D show representative real-time perfusion plots. 6A isvehicle-treated male; 6B is a GRI therapy treated male; 6C is avehicle-treated OVX-female; and 6D is a GRI therapy treated OVX-female.

FIGS. 7A-7B show analyses of males and OVX-females treated with vehicleor GRI therapy. 7A shows TTC-staining of fresh brain tissue and 7B showsinfarct volume analysis.

FIGS. 8A-8B show representative LSCI images and real-time perfusioncurve during ischemia (8A) and reperfusion (8B), in the transient MCAocclusion (tMCAo) model of stroke and restoration of CBF.

FIGS. 9A-9D show representative real-time electrogram traces for micetreated with combinations of vehicle or GRI and RIC-mock or RIC therapy(RIC-Thr).

FIG. 10A-10B analyses of mice treated with combination vehicle or GRIand RIC-mock or RIC therapy. 10A shows a plot of the linear regressionand 10B shows reoxygenation of brain tissues (PbtO2) for each treatmentcombination.

FIGS. 11A-11D show inflammatory gene expression in brains of micetreated with combinations of vehicle or GRI and RIC-mock or RIC therapy(N=6/gp). 11A shows measurement of iNOS expression; 11B showsmeasurement of iCAM; 11C shows measurement of IL-1β; and 11D showsmeasurement of IL-6.

FIGS. 12A-12D show that GRI therapy enhanced the neuroprotectiveefficacy of RIC therapy in reperfused tMCAo stroke model instreptozotocin (STZ)-induced diabetic mice. 12A shows % infarct volumefollowing treatment, and 12B shows % swelling, and 12C shows infarctvolume in GRI+RIC combination therapy group, vehicle-mock operatedcontrol, GRI therapy singly and RIC therapy alone. 12D showsrepresentative TTC-stained fresh brain tissue images wherein GRI therapyin combination with RIC therapy enhanced neuroprotection in the tMCAomodel with the comorbidity of diabetes.

FIGS. 13A-13B show analyses of mice treated with vehicle, GRI, IVT orGRI+IVT. 13A shows stroke injury/edema volume quantification. 13B showsrepresentative coronal slices from the MCA region.

FIGS. 14A-14B show neurological deficit scoring (NDS) and survival. 14Ashows NDS based on a modified Bederson Scale performed for all survivingmice at 96 h post-eMCAo in treatment groups of vehicle, GRI, IVT orGRI+IVT. 14B shows a Kalan-Meier survival curve for all 4-groupsfollowed for 2-weeks.

FIGS. 15A-15B show tests for long-term functional outcomes for micetreated with vehicle, GRI, IVT or GRI+IVT. 15A shows recovery ofmuscular-strength (hanging wire test) and 15B shows protection oflearning-memory function (novel object recognition test).

FIG. 16 shows representative series of fixed brain samples from micetreated with vehicle, GRI, IVT or GRI+IVT at day-15 post-eMCAo.

DETAILED DESCRIPTION

The following descriptions and examples illustrate some exemplaryembodiments of the disclosed invention in detail. Those of the skill inthe art will recognize that there are numerous variations andmodifications of this invention that are encompassed by its scope.Accordingly, the description of a certain exemplary embodiment shouldnot be deemed to limit the scope of the present invention.

The invention is a method of treatment for stroke and, in particular, itis a treatment for stroke in a subject or patient who has at least onecomorbid condition. This is a patient population that is particularlypredisposed to stroke, with comorbid conditions that complicate medicalmanagement of conventional treatments, and will be likely treated withRIC, EVT, IVT and/or any possible combinations of these 3 therapies Themethod of the invention enhances and preserves endogenous —SNO, abioactive form of NO that improves microvascular flow duringhypoxia/ischemia without altering blood pressure. A key benefit of theinvention is that it extends the effective safe window of thrombolysiswith intravenous tissue plasminogen activator (IVT) therapy beyond thecurrent 3 hours post-onset of stroke when the risk of hemorrhage/HT iselevated and IVT is not approved beyond this window of 3 hours,particularly in stroke patients with comorbidities.

In one embodiment, the invention is a method of treatment for a strokein a subject in need thereof, comprising administering a therapeuticallyeffective amount of an GSNOR inhibitor, wherein the therapeuticallyeffective amount is sufficient to inhibit brain tissue injury, as anadjunct therapy with RIC therapy. Known GSNOR inhibitors include N6022,cavosonstat, N91115 and N6338. Any of these inhibitors are suitable forthe method of the invention. The GSNOR inhibitor may be administeredprior to performing RIC therapy or it may be administered concurrentlywith the RIC therapy. The RIC is performed according to an FDA-approvedprotocol and/or by using an FDA-approved device.

In another embodiment, the method further comprises the step ofadministering a therapeutically effective amount of IVT, wherein thetherapeutically effective amount is sufficient to achieve thrombolysis,and/or administering EVT for clot retrieval. The method is surprisinglyeffective even when the “window of opportunity” (e.g., 3 hrs) has passedsince a suspected time of onset of the stroke and the chance ofhemorrhage would otherwise by increased.

In another embodiment, the subject is suspected to be suffering from astroke or is expected to have higher brain injury and worsened outcomes(such as stroke with diabetes, hypertension etc.) and/or hemorrhagictransformation. Stroke in subjects having comorbidities is particularlydangerous and likely to result in exacerbated brain injury worseningoutcomes due to the comorbid condition(s). In particular, the treatmentmay be administered prophylactically if a stroke or higher brain injuryor hemorrhagic transformation appears imminent when a patient alsosuffers from at least one comorbid condition.

The method of the invention comprises a combination therapy that wassurprisingly effective in patients whose symptoms are resistant toconventional treatments. This resistance arises from the complexunderlying conditions of comorbidity, such as, type II diabetes,hypertension, cardiac disease, coronary artery disease,arteriosclerosis, atherosclerosis, myocardial infarct, congestive heartfailure, peripheral vascular disease, dementia, cerebrovascular disease,chronic pulmonary disease, congestive obstructive pulmonary disease,kidney disease, kidney failure, chronic liver disease and metabolicsyndrome. A patient with even one of these conditions is frequentlyrefractory to all currently available treatments procedures, andpresents a higher risk of complications and poor outcomes, both from thestroke itself and/or procedures to treat stroke. In one embodiment, thesubject has one of these comorbid conditions, and in other embodiments,the subject has many of these comorbid conditions.

In one embodiment, the subject is suffering from the comorbid conditionsof diabetes and hypertension, and the GSNOR inhibitor is administered asan adjunct therapy to RIC therapy. Optionally, IVT and/or EVT are alsoadministered to dissolve and/or remove a thrombus or blood clot from anartery, thereby restoring perfusion of the brain region in which bloodflow was being blocked by the thrombus/clot. As a result of thetreatment, ischemic injury and brain tissue infarct can be reduced,minimized or avoided, and functional activity that would otherwise beimpaired is preserved or restored, at least in part.

In one embodiment, the invention is a method of restoring brainmicrocirculatory flow in a subject suffering from at least one comorbidcondition, comprising the step of administering in combination with RICtherapy and a therapeutically effective amount of an GSNOR inhibitor,wherein the therapeutically effective amount is sufficient to allowreperfusion of a brain region wherein the brain microcirculatory flowwas previously blocked. The GSNOR inhibitor may be administered prior toinitiation of the RIC therapy, or the GSNOR inhibitor may beadministered concurrently with RIC therapy.

In another embodiment, the invention is a method of restoring brainmicrocirculatory flow in a subject suffering from at least one comorbidcondition, comprising the step of administering in combination atherapeutically effective amount of an GSNOR inhibitor, wherein thetherapeutically effective amount is sufficient to allow reperfusion of abrain region wherein the brain microcirculatory flow was previouslyblocked, and RIC therapy, and these are followed by administration ofIVT for thrombolysis and/or EVT for clot retrieval.

As used herein, the term “GRI therapy” refers to administration ordelivery of a GSNOR inhibitor.

As used herein, the term “RIC therapy” refers to administration ordelivery of remote ischemic conditioning (in regimens of pre, per and/orpost conditionings), a medical procedure that aims to reduce theseverity of ischemic injury to an organ such as the heart or the brain,most commonly in the situation of a heart attack or a stroke, or duringprocedures such as heart surgery when the heart may temporary sufferischemia during the operation, by triggering the body's naturalprotection against tissue injury. The procedure involves repeated,temporary cessation of blood flow to a limb to create ischemia in thetissue. This “conditioning” activates the body's natural protectivephysiology against reperfusion injury and the tissue damage caused bylow oxygen levels. It typically can be administered or performed using asimple blood pressure cuff, or it can be performed using a devicedesigned for this purpose, such as one currently in review at the FDA.

As used herein, the term “intravenous tissue plasminogen activatortherapy” or “IVT” refers to a medical procedure involving administrationof an FDA-approved intravenous thrombolytic (IVT). Tissue plasminogenactivator (tPA), is an intravenous medicine given during ischemicstroke, i.e., in a stroke caused by a blood clot, wherein IVT candissolve the stroke-causing clot. Studies have shown that certain strokepatients who receive tPA within 3 hours, have better outcomes. IVT iscurrently FDA-approved for use only within the initial 3 hours window ofpost-ictus.

As used herein, the term “endovascular thrombectomy” or “EVT” refers toa medical procedure for mechanical removal of a thrombus (blood clot)under image guidance using a catheter type device. A thrombectomy ismost commonly performed for an arterial embolism, which is an arterialblockage often caused by atrial fibrillation, a heart rhythm disorder,blood clot etc.

As used herein, the term “biopharmaceutical compound” may be considereddifferent from a “synthetic pharmaceutical compound”, and may include anon-immunogenic adeno-associated virus carrying a transgene togenetically intervene and inhibit GSNO-reductase for long-term benefitsvia genetic intervention.

As used herein, the term “pharmaceutically acceptable” means anymolecular entity or composition that does not produce an adverse,allergic, or other untoward or unwanted reaction when administered to anindividual. As used herein, the term “pharmaceutically acceptablecomposition” is synonymous with “pharmaceutical composition”. Apharmaceutical composition of the invention may be used for human andveterinary applications. In a preferred embodiment of the invention, acomposition of the invention is administered to humans, most preferablyto children. The pharmaceutical compositions of the invention may beadministered to an individual alone, or in combination with other activeingredients.

The invention is a method of treatment to enhance and preserveendogenous —SNO, a bioactive form of NO that improves microvascular flowduring hypoxia/ischemia without altering blood pressure (Stamler et al.Science 1997, 276(5321):2034-2037; Shu et al. Cell Mol Life Sci 2015,72(23):4561-4575). Thus, the invention is a method of treatment ortherapy to protect against stroke injury and improve benefits of RICtherapy in stroke with comorbidity. Of interesting note here, —SNO, theendogenously preserved vasoactive NO, s-nitrosylates tPA to enhance theeffects of IVT in stroke treatment (Stamler et al. Proc Natl Acad SciUSA. 1992, 89(17):8087-8091). GSNOR is an endogenous enzyme and themaster regulator which balances the endogenous level of —SNO bydegrading/reducing GSNO (Jahnova et al. Plants (Basel). 2019; 8(2)).Thus, an increased activity of GSNOR may deplete endogenous NO levelduring stroke and therapies such as RIC therapy. Without being bound bytheory, it is believed that the method of the invention counteracts thisdepletion of endogenous NO that is caused by increased GSNOR activity.

Acute ischemic stroke, often due to TE occlusion, is the most common(˜87%) type of strokes. Microcirculatory reflow after stroke isessential for the reoxygenation of ischemic penumbra and to preventinfarct progression. Despite reperfusing “large” vessels with theFDA-approved stroke therapies, reoxygenation of brain frequently remainsincomplete, and hence, a critical barrier to improve post-strokeoutcomes. Microcirculation is notably enhanced with freely diffusiblevasculo-humoral NO, which improves tissue perfusion during hypoxia;however, a recent clinical trial concluded no benefits from aconventional NO3⁻/NO2⁻ therapy in stroke. Moreover, a risk of bloodpressure BP lowering remains warranted with the long-term use ofexogenous NO-therapy. Therefore, an objective of the invention is theenhancement and preservation of “endogenous” NO-metabolome formicrovascular protection in stroke.

Endothelial NO synthase (eNOS) activity prominently constitutes thevascular NO-pool that enhances microcirculation. Genetic impairment ordeletion of eNOS in mice augmented microvascular dysfunction and braintissue hypoxia during ageing and also exacerbated injury after acutestroke; thus, the critical role of endothelial NO was established inmicrovascular perfusion and resultant neuroprotection. Following injury,eNOS expression decreases in micro-vessels and activity is concomitantlyimpaired; however, NO generated or delivered remotely was foundtransportable to a distant ischemic organ resulting in improvedmicrocirculation via hypoxic vasodilation. Thus, myocardialoverexpression of eNOS protected against ischemia-reperfusion (IR)injury in liver in an experimental model. This mechanism of NOpreservation and carriage also involvess-nitrosylation/trans-nitrosylation of thiol group of proteins(—SH>—SNO) such as glutathione (GSH) and hemoglobin (Hb) within redblood cells; thereby, s-nitrosylated GSH (GSNO) and Hb (HbSNO) wererecognized as the major endogenous chariots of bioactive NO whichprotect against hypoxia and IR-injuries. Intravenous GSNO-therapy andincreased HbSNO with NO-inhalation improved outcomes in stroke models.Furthermore, —SNO-therapy reduced embolization in human patients,attenuated secondary ischemia after sub-arachnoid hemorrhage, and ofparticular interest, induced hypoxic vasodilation to enhance cerebralblood flow in rodents; however, a risk of increased injury remainsassociated at certain doses of exogenous GSNO in stroke.

The enzyme GSNO reductase (GSNOR or ADH5) is a class III alcoholdehydrogenase which degrades —SNO/GSNO. Genetic deletion andpharmacological inhibition of GSNOR protected against IR-injury due toincreased endogenous level of —SNO. A GSNOR inhibitor preservedvasculo-protective benefits of low-level endogenous NO in hypertension,one of the most common comorbidities associated with ageing and stroke.GRI therapy, in hypertensive rats with impaired NO production, preservedflow-mediated dilation and protected against microvascular and conduitartery dysfunction. GRI therapy also protected against mechanicalreperfusion injury in brain; however, the effect of stroke on GSNORexpression/activity particularly in a clinically relevant TE-strokemodel has been previously unknown, as have any potential benefits of itsmodulation in different stroke models, ages, sexes and reperfusiondynamics. The examples of the invention will demonstrate theeffectiveness of the methods disclosed herein.

Vasculo-humoral NO is decreased during acute stroke in both, rodents andhuman patients. Others and we have reported that therapies capable ofmodulating endogenous NO-metabolome, such as RIC, enhances cerebralblood flow, protects against ischemic brain injuries, and improvedefficacies of late IVT in stroke. Of note, NO following s-nitrosylationof tissue plasminogen activator (tPA) promotes thrombolysis. Herein, theexamples of the invention will further demonstrate that TE-strokeupregulates GSNOR expression/activity, which in turn, will attenuateendogenous —SNO level and augment stroke injury. Furthermore, examplesof the invention demonstrate that GRI therapy in stroke will preserveendogenous —SNO to augment microcirculation, improve benefits ofreperfusion to reoxygenate brain and attenuate injury to improvefunctional outcomes.

GSNOR inhibitors are known and have been used in investigational studiesof ischemia and reperfusion. For example, Khan et al. (Brain Res. 2020Aug. 15; 1741:146879.) teaches that mice were treated with N6022 (5mg/kg) at 2 hr post ischemia/reperfusion, daily for 3 days or 2 weeks.Neurological score, survival rate and motor/cognitive skills wereimproved. However, these results were not performed in clinicallyrelevant thromboembolic stroke model nor were they validated indifferent stroke models. While Khan mimics conditions of a specificminor population of stroke patients reperfused following mechanical EVT,EVT alone does not guarantee microvascular perfusion and brain tissuereoxygenation. Furthermore, Khan fails to teach or suggest IVT, RIC, orany combination of any adjuvants with RIC/IVT. Landman et al. (StrokeVol 50; 7, 2019 p 19534-1939) teaches the hypothesis that RIC inducesthe release of humoral factors including NO, which activate afferentneural and humoral pathways and shows that RIC reduces oxidative damageand suppresses inflammatory responses in the brain. However, Landmanfails to make any connection of RIC to other therapies or considersubjects having comorbidities.

The GSNOR inhibitor may be administered via any suitable route,including but not limited to intravenous, intranasal, nebulization,oral, intramuscular, caudal, intrathecal, and subcutaneous. Compositionscomprising GSNOR inhibitors include any pharmaceutically-acceptablecarrier or buffer suitable for administration to a subject. In apreferred embodiment, the pH of the composition is in the range of 6.5to 8.2 or 6.5 to 8.0 or 6.5 to 7.0. In another preferred embodiment, thepH of the composition is in the range of 6.6 to 8.2 or 6.6 to 8.0 or 6.6to 7.0. In a preferred embodiment, the pH is 6.6, 6.7, 6.8 or 6.9. In amore preferred embodiment, the pH is 6.7 or 6.8. In a further preferredembodiment, the pH is 6.8. In another more preferred embodiment, the pHis 7. In a particularly preferred embodiment, the pH is 6.8 and thebuffer strength is 0.05 M. In another particularly preferred embodiment,the pH is 7.0 and the buffer strength is 0.02 M. Various buffers may beused to prepare a pharmaceutical composition of the invention, providedthat the resulting preparation is pharmaceutically acceptable. Suchbuffers include, without limitation, acetate buffers, citrate buffers,phosphate buffers, neutral buffered saline, phosphate buffered salineand borate buffers. It is understood that acids or bases can be used toadjust the pH of a composition as needed. In a preferred embodiment, thebuffer is sodium citrate buffer. In a more preferred embodiment, thesodium citrate buffer comprises tri-sodium citrate, citric acid andpurified water. In another preferred embodiment, the buffer is phosphatebuffer. Preferably, the buffer strength is in the range of 0.01 to 0.1 Mor 0.01 to 1 M. More preferably the buffer strength is 0.01 M to 0.06 M.In a further preferred embodiment, the buffer strength is 0.02 M to 0.06M, more preferably 0.02 M to 0.05 M and most preferably 0.02 M or 0.05M. The active ingredients and/or excipients can be soluble or can bedelivered as a suspension in the desired carrier or diluent. In apreferred embodiment of the invention, the liquid pharmaceuticalcomposition is a solution.

Before exemplary embodiments of the present invention are described ingreater detail, it is to be understood that this invention is notlimited to any particular embodiments described herein and may vary. Itis also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Where a range of values is provided, it is understood that eachintervening value between the upper and lower limit of that range (to atenth of the unit of the lower limit) is included in the range andencompassed within the invention, unless the context or descriptionclearly dictates otherwise. In addition, smaller ranges between any twovalues in the range are encompassed, unless the context or descriptionclearly indicates otherwise.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Representative illustrativemethods and materials are herein described; methods and materialssimilar or equivalent to those described herein can also be used in thepractice or testing of the present invention.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference, and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual dates of publicavailability and may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as support for the recitation in the claims of suchexclusive terminology as “solely,” “only” and the like in connectionwith the recitation of claim elements, or use of a “negative”limitations, such as “wherein [a particular feature or element] isabsent”, or “except for [a particular feature or element]”, or “wherein[a particular feature or element] is not present (included, etc.) . . .”.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

EXAMPLES

The following Examples provide exemplary designs and methods forfabricating and using microgrippers of the invention. These Examplesdescribe materials and methods for using embodiments illustrated inFIGS. 1-16. Additional details can be found in the section entitled“Brief Description of the Drawings”.

The following Examples of the invention are provided to demonstrate thatTE-stroke enhances GSNOR expression/activity, which in turn, willattenuate endogenous —SNO level and augment stroke injury. The Examplesalso demonstrate that GRI therapy for stroke will preserve endogenous—SNO to augment microcirculation, improve benefits of reperfusion toreoxygenate brain and attenuate injury to improve functional outcomes.

Models of Stroke

The following models of stroke were used in the Examples of theinvention to demonstrate methods of treatment.

The eMCAo Model of TE-Stroke

Human fibrinogen-supplemented clot was prepared to induce TE-stroke asreported by Hoda et. al. Briefly, under isoflurane anesthesia, the rightcommon carotid artery (CCA), external carotid artery (ECA) and internalcarotid artery (ICA) were exposed with blunt dissection. A modifiedmicrocatheter containing a 9.0±0.5-mm long clot was inserted into theright ECA, advanced into the ICA and the clot was gently delivered intothe MCA region along with 100-μL of 1× sterile phosphate buffered saline(PBS). The catheter was retracted, the ECA-arteriotomy was ligated toprevent bleeding, and wounds were sutured. Sham-operated mice underwenta similar procedure including infusion of 100 μL of 1× sterile PBSwithout a clot. Mice were returned to clean warm recovery cages untilconscious and were given free access to NAPA-gel and chow diets alongwith water ad-libitum until sacrifice.

The Photothrombotic (PT)-Model of Stroke Aged mice (16±1-mo) weresubjected to PT-stroke via a novel lateral side surgical approachwithout craniotomy resulting in the thrombotic occlusion of distal maintrunk of MCA. For the purpose, mice were anesthetized with isofluorane,prepared for surgery, and rose Bengal dye (50 mg/kgbwt in 200 uL of 1×sterile PBS) was intravenously (IV) infused. Mice were immediately puton a robotic stereotaxic frame, the head was rotated to face right sideup, and the skull was exposed. The distal trunk of MCA was visualizedunderneath the skull bone (without craniotomy) using a 532 nm lasersource (8-mm beam diameter; 4.5 mW) integrated to an ID8-iris (ThorLabs,NJ, USA). The MCA region above the zygomatic arc was exposed with thelaser for 20-min from a distance of ˜2-3-mm. Following 20-min laserexposure, thrombotic occlusion of the distal trunk of MCA was confirmedusing laser doppler flowmeter showing >60% drop in CBF and finally thelateral side skin wound was closed.

The pMCAL Model of Stroke

The procedure for pMCAo was performed in mice as reported by Hoda et al.(Stroke 2012, 43(10):2794-2799). Briefly, under anesthesia, the junctionof left zygomatic arch and squamous bone was drilled to make a 2-3 mmburr hole to expose the main trunk of MCA. The left MCA distal to thelenticulostriate branches was electrocauterized carefully withoutbleeding. The wound was closed, and mice were put under post-op recoveryas described above. Sham-operated group underwent similar surgicalprocedures but without electrocauterization.

The Reperfused tMCAo Model of Stroke

The tMCAo surgical procedure is similar to TE-stroke surgery, however,the transient occlusion is achieved using a suture instead of a clot asreported by Hoda et al (supra). Briefly, a 6-0 monofilament suture with˜3-mm silicone-coated tip designed to induce tMCAo in mice (Doccol Corp,USA) was introduced into the ICA via an ECA stump, directed and gentlyadvanced into the MCA region until the suture was wedged to occlude theproximal stem of MCA. The occlusion was maintained prior to withdrawingthe filament after 45 minutes, the ECA-arteriotomy was ligated and thereperfusion was allowed following suture withdrawal. The sham-operatedgroup underwent similar procedures without occlusion, and mice recoveredas above.

Materials and Methods

Animal Models and Experimental Approach

All mice under C57/B6 background were bred, housed, used and relatedexperimental procedures were performed in accordance with the approvedprotocol of Institutional Animal Care and Use Committees (IACUCs) of St.Joseph's Hospital and Medical Center (SJHMC), and Augusta University,Augusta, Ga. Effect of stroke on GSNOR expression/activity was firstdetermined in a clinically relevant partially-humanized TE-stroke,embolic middle cerebral artery occlusion (eMCAo) model in aged(16±1-mo-old) male mice. Next, an effective dose-finding study of GRItherapy (stroke injury size) was performed in aged (16±1-mo-old) malemice in a photothrombotic (PT) model of stroke with the reduction ininfarct volume as the primary outcome. The safety and benefits of thebest dose of GRI therapy in reducing infarct volume as the primaryoutcome was also confirmed in reproductively senescent aged(16±1-mo-old) female mice subjected to PT-stroke. To test the benefitsof GRI therapy during permanent ischemia, GRI was also tested in adult(14±1-weeks) mice of both sexes subjected to permanent model of middlecerebral artery ligation (pMCAL). All females were ovariectomized at theage of 12-weeks, i.e., 2-weeks prior to stroke surgery. A 2×2 factorialdesign was adopted [2 Sexes (MALE vs. FEMALE)×2 GRI (NO vs. YES)] suchthat all 4 groups of both sexes were subjected to pMCAL followed byrandomization into two different treatment cohorts: GRI therapy or equalvolume of Vehicle treatment. All mice were tested for behavioral outcomeon a narrow beam test, relative CBF measurement using laser specklecontrast imaging (LSCI) and infarct volume size. Next, benefit of GRItherapy was tested with and without RIC therapy in preserving benefitsof endogenous NO in a reperfused stroke model in diabetic male mice(14±1-weeks). RIC therapy has been reported to enhance CBF in strokemodels likely via enhancing endogenous NO but remains untested incomorbid (such as diabetic) stroke, a patient population which are leastbenefited from the FDA-approved reperfusion therapies such asendovascular thrombectomy (ET). To mimic a clinical ET-like scenario,GRI therapy was tested in a reperfused transient MCA occlusion (tMCAo)model of stroke. A 2×2 factorial design was adopted [2 GRI (NO vs.YES)×2 RIC (NO vs. YES)] such that all 4 groups were subjected to tMCAofollowed by randomization into 4 different treatment cohorts: Vehicle,GRI, RIC and GRI+RIC. Outcome measures such as brain tissue oxygenation(PbtO2), acute inflammatory genes response, neurological deficit, edemaas percent (%) swelling and infarct volumes were assessed. Lastly, GRItherapy was also tested with and without late-IVT, an FDA-approvedthrombolytic reperfusion therapy, in eMCAo for which again a 2×2factorial design was adopted [2 GRI (NO vs. YES)×2 IVT (NO vs. YES)] asabove: Vehicle, GRI, IVT and GRI+IVT groups. Adult male mice(16±2-weeks) were used in this experiment.

Treatments after Stroke

Briefly, different doses of GRI therapy (N6022; Axon Medchem, USA) atvarious time points, as indicated for each of the following Examples,were tested in different sets of experiments and stroke models. Ingeneral, GRI therapy was IV-infused via the tail vein following stroke,and IP-repeated after stroke as indicated. RIC and IVT therapies wereperformed as indicated. All control groups were either infused withequal volume of vehicle (for GRI- and IVT-therapies) or underwentRIC-mock for sham-operation of RIC therapy.

Laser Doppler Flowmetry (LDF) and Laser Speckle Contrast Analysis(LASCA) Imaging

Cortical LDF (Perimed Inc, Sweden) was performed to confirm theinduction of stroke in anesthetized mice. Briefly, mice wereanesthetized as above and a shallow indent was made in the parietalskull using a low-speed drill to place the LDF probe holder (PH07-6,Perimed Inc) aligning the holder hole at the stereotaxic coordinates(AP: 2-mm and lateral 3-mm with respect to bregma). The needle ofLDF-probe (PH407, Perimed Inc) was inserted into the probe holder andthe signal was recorded to confirm a significant drop in CBF followingstroke. Mice were anesthetized as above, body temperature was maintainedat 37±0.5° C., the skull was shaved, and a midline skin incision wasmade to primarily expose the MCA region. Perfusion images were acquiredusing PeriCam high resolution LASCA-Imager (PSI-Z system, Perimed Inc.)with a 70-mW built-in laser diode for illumination and 1388×1038 pixelsCCD camera installed 10 cm above the skull (speed 19 Hz, and exposuretime 6 mSec, 1.3×1.3 cm). Acquired video and images were analyzed forthe dynamic changes in CBF. Overall perfusion of the ipsilateralischemic hemisphere was determined, normalized with the equal size ofregion of interest (ROI) from the uninjured contralateral hemisphere andthe relative CBF was calculated.

MRI Acquisition

All MRI experiments were conducted using a 7-Tesla horizontal magnetwith a clear bore of 20-cm in diameter interfaced to a Bruker Avanceconsole. Anesthetized mice underwent the following MRI-fast spin echo(RARE factor=8) with effective echo time of 47 ms to createcorresponding T2-weighted images to determine absolute edema/strokeinjury volume. Ex vivo diffusion tensor imagining (DTI) was performedwith overnight MRI of fixed brains through a pulse-gradient spin echosequence to determine the diffusion parameters including apparentdiffusion coefficient (ADC), tensor trace and fractional anisotropy(FA).

MRI Data Analysis

Image post-processing for edema was performed using ImageJ coupled within-house designed ImageJ macro scripts. T2-weighted images were used todetermine the volume of cerebral edema by drawing an irregular ROI toencircle the regions exhibiting edema in each impacted image slice. Theimpact volume was determined by summation of the ROIs in all theimpacted image slices and then multiplying with the thickness of theimage slice. DTI analysis was done using the vendor-supplied softwareParaysion 5.1 (Bruker Inc.), from which the associated diffusionweighted, FA and ADC images were generated.

Analysis of GSNOR Expression and Activity after Stroke

Plasma was isolated from freshly drawn blood after cardiac punctureunder deep isoflurane anesthesia, and mice were sacrificed to collectbrain tissue. Plasma and brain tissue samples were snap-frozen untilassay.

Immunoblot Assay for GSNOR Expression

Gel electrophoresis followed by immunoblot for the GSNOR expression inbrain tissue samples (perfused with 50-mL of chilled 1× sterile PBS) wasperformed using conventional methods. Briefly, brain homogenates inmodified RIPA buffer (Upstate, Lake Placid, N.Y.), supplemented with40-mM NaF, 2-mM Na₃VO₄, 0.5-mM phenylmethylsulfonyl fluoride and 1:100(v/v) of proteinase inhibitor cocktail (Sigma), were electrophoresed andimmunoblotted separately against antibodies for anti-GSNOR (Sigma), andanti-beta actin (β-Actin; Santa Cruz Biotech, USA) as loading control.Immuno-densitometric signal for GSNOR was quantified in arbitrary units(AU) using ImageJ NIH free software and was normalized with thecorresponding expression of β-Actin.

GSNOR Activity Assay A method of GSNO dependent NADH consumption wasused to determine GSNOR activity as reported. Briefly, brain tissue washomogenized in a buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl,1-mM EDTA, 0.1% Triton X-100 and 1:100 protease inhibitor cocktail andcentrifuged (10,000 g×10 minutes at 4° C.) to obtain supernatant. Next,plasma and brain tissue supernatant were diluted to a proteinconcentration of 1 mg/mL and 0.1 mg/mL, respectively, in a reactionbuffer containing 20 mM Tris-HCl (pH 8.0), 0.5 mM EDTA and 0.7504 NADHfollowing which samples were incubated in triplicate with or without 100μM GSNO in 96-well plate. The consumption of NADH was monitored withfluorescence spectrophotometry at an excitation wavelength of 340 nm andemission at 460 nm. GSNOR activity was calculated by subtracting therate of NADH consumption incubated in the presence of GSNO minus therate of NADH consumption without GSNO in samples.

Euthanasia and 2,3,5-Triphenyltetrazolium Chloride (TTC) Staining

TTC-stain differentiates between metabolically active (live or penumbra)and inactive (dead or core) tissues after stroke. TTC is a white powderand the solution remains colorless, which is reduced to red1,3,5-triphenylformazan (TPF) by the enzymatic action of variousdehydrogenases primarily mitochondrial dehydrogenase from the livingtissues, while the core remains as white. Therefore, larger white areaindicates higher injury and infarction volume. After performing ATT,mice were overdosed and deeply anesthetized with high isofluorane (5%).Brains were very quickly perfused with cold 25 ml of 0.01 Mphosphate-buffered saline (PBS), harvested fresh and immediately placedin a metallic mouse brain matrix. Looking at the infarcted area, 5blades were placed in alternate gaps to cut and obtain 2-μm×4 coronalslices. Sections were individually placed in a 35-mm dish containingpre-warmed (37° C.) 3-ml of 5% TTC in PBS (Sigma) for 20-25 minutes at37° C., followed by 2× washing with cold PBS and fixation with 10%formalin. In order to image, fixed sections were taken out of the dishand placed in order on a high-resolution Cannon Scanner. After scanning,images were cropped and saved for analysis. Corrected infarct volume wasestimated using gray scale image and Scion Image software and presentedas the Corrected % infarct volume normalized to the uninjured side.

Behavioral Tests

Beam Walk Test for Motor Balance and Coordination

Motor balance and coordination test was performed using the narrowbalance beam walk. The beam apparatus consisted of a graduated beam, wasfixed 20-cm above the tabletop on two poles (6-mm flat width×125-cmlong). A black box was fixed at the end of narrow beam as the finishpoint containing nesting (bedding) material from the home cage toattract the mouse. A lamp (with 60-watt light bulb) is used to shinelight above the start point, serving as an aversive stimulus. Mice weretrained to walk on the barrow beam prior to injury and before performingthe post-stroke test. During training, mice were encouraged to keepmoving across the beam by gently poking or pushing on their back.Training trials were repeated for 3 trials×3 consecutive days or untileach animal crosses the beam 3× without stopping or turning around, andwithout any assistance. On a final day of testing, each mouse was testedto walk 100-cm on the beam with 3-trials×5 min interval between trials.Performance of each mouse on the narrow beam was quantified by measuringthe time it took for the mouse to traverse the 100-cm distance on thenarrow beam and the number of foot slips that occurred in each trial.The mean value of 3 trials was calculated for the time taken intraversing the 100-cm distance on the beam, and the number of footfaults.

Neurologic Deficit Score (NDS)

Neurologic deficits after stroke in mice were assessed by blindedinvestigators blinded on a 5-point modified Bederson NDS-scale. Thehighest number indicates the worst outcomes while lower number indicatesbetter neurological outcomes: 0, no deficit (normal mice); 1, forelimbflexion deficit on contralateral side; 2, flexion deficit along withdecreased resistance to lateral push and torso-turning to theipsilateral side when held by tail; 3, all deficits as in Score 2including very significant circling to the affected side during the moveinside the cage and reduced capability to bear weight on the affectedside; 4, all deficits as above but rarely willing to move spontaneouslyand preferring to stay at rest.

Hanging Wire Test for Forelimb Coordination and Muscular Strength

This test was used as a measure of grasping ability, forelimb strengthand muscular coordination movements. The apparatus is consisted of a55-cm long 1-mm diameter single metal cord stretched firmly between twometal stands, 50-cm above a standard mouse cage filled with soft paperbedding material. In the training process, mice are gently held andtaken closer to the wire to grab it with their two forelimbs. Mice weretrained for 3 trials×3 consecutive days before the actual test, or untilthey are trained to grab it with two forelimbs. For the actual test, thelatency to lose the grip on the wire falling into the cage was recordedfor 3 trials×5-min interval between trials for each mouse.

Novel Object Recognition (NOR) Test for Cognitive Function

Object recognition memory is one of the domains of cognition that isoften impaired in aged (non-demented) individuals as well as in patientswith other forms of dementia. Importantly, the rodent NOR-task has beendescribed as a model of (non-spatial) recognition memory. This form ofmemory is believed to consist of a recollected (episodic) and afamiliarity component, i.e., behaviors that are demonstrated in theNOR-task when subjects explore a novel object more than a familiar one.The NOR-task has become a popular method for rodent studies withparticular relevance to dementia and neuropsychiatric disorders.

Behavioral assessment by NOR test that reliably evaluates the nonspatialworking memory of the subcortical region was performed 4-wks afterstroke. Briefly, mice were habituated in an activity box andfamiliarized with two identical objects placed at a set distance apart.On the day of trial, each mouse was individually given similar trial andthe time spent (T_(f)) with the familiar object was recorded. After thefamiliar object trial, the mouse was then removed from the environmentfor a set amount of time and one of the two previously used (familiar)objects was replaced with a novel object that was different from thefamiliar object in shape, texture, and appearance. The time spent(T_(n)) by the mouse with the novel object upon exposure during theprobe trial was then recorded in one 5-min trial. This test is based onthe natural tendency of mouse to investigate a novel object rather thana familiar one, which reflects the use of learning and recognitionmemory processes. The capability of the mouse to discriminate between afamiliar vs. novel object was determined as the discrimination index,which is calculated by DI=(T_(n)−T_(f))/(T_(n)+T_(f)). A lower DI-valuereflects poor cognitive function, while a higher DI-values demonstratesa better learning and memory function.

Data Analysis

Data and Statistical Analysis

Quantitative data were presented as mean±standard deviation (SD).Wherever needed, unpaired t-test was performed to determine statisticalsignificance. A log transformation was used prior to statisticalanalysis as needed to stabilize variance across groups. Non-normal databetween groups were compared using nonparametric asymptotic two-sidedWilcoxon rank sum test. In experiments with 2×factorial design, afactorial analysis of variance (ANOVA) with interaction between twotherapies followed by post-hoc comparisons using independent t-tests wasused to analyze the outcome measures. Bonferroni multiple correctionswere applied in the post-hoc analysis. In the absence of a significantinteraction, the main effects were considered to be additive whencombined. A p-value <0.05 was considered statistically significant. Allstatistical analyses were conducted using STATA 15.

Data

FIG. 3E, FIG. 4B, FIG. 4D, FIGS. 6A-D, FIG. 8B, FIG. 9A-D, and others inthis description are real time curves/plots from the instrument. Theyare presented to show the real time output as an example from oneanimal. For each animal subject, the values are averaged and calculatedas either a percentage (%)(e.g., for CBF/perfusion) or in mmHg foroxygen (after converting the millivolt output) and then they are plottedas the data for each group.

Example 1

Effect of eMCAo on GSNOR protein expression and activity.

The eMCAo model was chosen because it is the most clinically relevantrodent model of TE stroke. Aged (16±1-mo) mice were subjected toeMCAo/stroke using a partially-humanized TE-clot. At 6 h post-eMCAo,blood was collected via cardiac puncture to isolate plasma and mice werequickly flushed-perfused with 25-mL of chilled 0.1M PBS prior tocollecting brain samples. Both plasma and brain samples were snap-frozenin liquid nitrogen until assays. FIGS. 1A and 1B show whole brain assayand the resultant relative normalized densitometry data confirmed thatthe protein expression of GSNOR was significantly (p<0.01) increased asearly as 6 h post-stroke as compared to sham-operated group. An activityassay based on the rate of GSNO-dependent consumption of NADH furtherconfirmed that the increase in protein expression of GSNOR translatedinto significant (p<0.001) acute increase in the enzymatic activity ofGSNOR as early as 6 h after stroke. FIG. 1C shows GSNOR activity inbrain and FIG. 1D shows GSNOR activity in plasma.

Example 2

Effect of GRI Therapy in Aged Mice of both Sexes following PT Stroke.

The PT stroke model was chosen because it is a thrombotic model ofmoderate injury. FIG. 2A shows a representative image of whole braincollected immediately after stroke to confirm the site (black arrow) ofocclusion in MCA region. FIG. 2B shows a Z-projection of a T2-weightedMR-image 48 h after stroke. The site of stroke injury is indicated witha white arrow. Different doses of GRI therapy or equal volume of vehicleto aged male mice (N=8/gp) given intravenously 1 h after PT-stroke (1,2.5, 5.0 and 10 mg/kg) showed a dose-dependent neuroprotective effect.However, a plateau in the neuroprotective effect was seen beginning fromthe GRI dose >2.5 mg/kg. Densitometry showing absolute infarct volume inFIG. 2C was performed on tissues sections, including those shown in FIG.2D. FIG. 2D shows representative TTC-stained set of coronal sections(2-μm thick) obtained from the whole brain from the aged male mice andstained freshly. GRI therapy significantly protected against the strokeand prevented the infarct/injury progression as compared to vehicletreated group (P<0.0001). Data are presented as Mean±SD. Statisticalsignificance was determined at P<0.05 and were indicated with asterisksor different letters indicating statistically different Means.

A sex difference was found in the basal level of plasma GSNOR activity,showing higher GSNOR activity in female mice compared to age-matchedmales (P<0.05; N=6/gp) as shown in FIG. 3A. Therefore, GRI therapy wasalso tested in aged female mice (N=10/gp). (F-H) The effective dose ofGRI therapy (2.5 mg/kg) for aged male mice was tested in aged femalemice as described above for males. As evident in FIG. 3B, there was asignificant increase (P<0.0001) in plasma GSNOR activity in agedfemales, too, after PT-stroke. GRI therapy after PT-stroke in femalessignificantly inhibited the post-stroke plasma GSNOR activity (P<0.01).GRI therapy was also effective in reducing the stroke injurysignificantly compared to vehicle treated group (P<0.05). FIG. 3D showsrepresentative TTC-stained set of coronal sections (2-μm thick) obtainedfrom the whole brain of aged female mice and stained freshly. Data arepresented as Mean±SD. Statistical significance was determined at P<0.05and were indicated with asterisks or different letters indicatingstatistically different Means.

Example 3

GRI Therapy Protects Against Permanent Ischemia Induced by pMCAoIndependent of Sex

Adult mice of both sexes (14±1-weeks; N=10/gp; females wereovariectomized, OVX) were subjected to pMCAL-stroke model withelectrocauterization leading to permanent occlusion of distal MCA trunk.Mice were anesthetized with isofluorane, prepared for surgery, and theright parietal bone of the skull was exposed. A craniotomy was performedto expose the distal part of MCA trunk and the artery was permanentlyelectrocauterized using a bipolar electrocautery. FIG. 4A shows arepresentative LSCI image. Immediately after stroke, a significant dropin CBF occurred. Mice were IV-treated with either GRI therapy (2.5mg/kg) or equal volume of vehicle at 1 h after stroke and the treatmentwas repeated daily for 2 days.

Mice were evaluated for behavioral outcomes at 72 h post-stroke. A 2sexes (Male vs. Female) by 2 treatments (GRI vs. Veh) ANOVA was used toanalyze results. All the data are expressed as mean±SD, pairs of meansindicated with different letters are significantly different (P<0.05).Narrow beam walk test determined that there was no significantdifference in the deterioration of motor function between two sexes inthe context of walking time to traverse through the beam length andnumber of foot slips. FIG. 4B shows the time to traverse the beam, andFIG. 4C shows the mean number of foot slips. GRI therapy was equallyeffective in improving motor function in both sexes.

Mice were also evaluated for CBF changes and infarct volume analysis at72 h post-stroke. A 2 sexes (Male vs. Female) by 2 treatments (GRI vs.Veh) ANOVA was used to analyze results. Changes in CBF showed a trendtoward slight differences in vehicle-treated OVX-females as evident fromthe plot shown in FIG. 4D.

At 72 h post-pMCAo, % CBF loss in the injured side of brain in bothsexes was evident, as shown in FIG. 5A for males, and FIG. 5B forOVX-females, but was also significantly higher in OVX-females comparedto males. GRI therapy improved restoration of CBF in both sexessignificantly compared to their corresponding vehicle treated groups.The GRI therapy treated groups in both sexes showed no significantdifference in % CBF when compared together, demonstrating that theeffect of GRI therapy in enhancing CBF remains sex independent.

Real-time perfusion was measured in mice at 72 h post-stroke. FIG. 6Ashows vehicle-treated males and Figure B shows GRI-treated males. FIG.6C shows vehicle-treated OVX-females (OVX-F) and FIG. 6D showsGRI-treated OVX-females. As with the % CBF and LSCI, vehicle-treatedOVX-females showed a slight trend towards difference in real-timeperfusion plots. At 72 h post-pMCAo, GRI therapy treated groups in bothsexes showed no significant difference in real-time perfusion plots whencompared together, demonstrating that the effect of GRI therapy inenhancing CBF remains sex independent.

TTC-staining of fresh brain tissue, shown in FIG. 7A, and infarct volumeanalysis, shown in FIG. 7B, further demonstrates that vehicle treatedOVX-females were significantly more prone to stroke injury compared totheir vehicle treated male counterparts. GRI therapy significantlyprevented the infarct progression sex-independently in both groups.

Example 4

GRI Therapy Improves Acute Brain Tissue Oxygenation followingReperfusion and Enhances Benefits of RIC therapy in STZ-induced DiabeticMice.

This example is a demonstration of reperfusion and restoration ofoxygenation (PbtO2) in reperfused tMCAo stroke model. Diabetes wasinduced in adult male mice (14±1 weeks of age) as confirmed after 7 daysof STZ-injection, followed by tMCAo stroke surgery at 3 weeks post-STZinjection. Stroke was induced with 60-min suture occlusion followed byreperfusion with suture withdrawal. GRI therapy or vehicle wasIV-infused 10-min prior to reperfusion and RIC therapy/RIC-mock wasperformed 1 h after reperfusion. A 2 GRI (GRI vs. Veh) by 2 RIC (RICtherapy vs. RIC-mock) ANOVA was used to analyze results. All the dataare expressed as Mean±SD, and pairs of Means indicated with differentletters are significantly different (P<0.05).

Representative LSCI, shown in FIG. 8A, and real-time perfusion curveduring ischemia and reperfusion, shown in FIG. 8B, demonstratedsuccessful induction of stroke and restoration of CBF. These resultswere comparable to those shown in Example 3 using the tMCAo model. PbtO2was measured at 6 h post-reperfusion (N=6/gp) using a 50-um thick oxygensensor integrated to an UniAmp multi-channel system (Unisense, Denmark).Measurement signal was averaged for 3 min during a period of stableoutput as shown in the representative real-time electrogram traces inFIGS. 9A-9D. FIG. 9A is representative of a mouse treated with vehicleand receiving mock RIC. FIG. 9B is representative of a mouse receivingvehicle and RIC therapy. FIG. 9C is representative of a mouse receivingGRI therapy and mock RIC. FIG. 9D is representative of a mouse receivingGRI therapy and RIC therapy. PbtO2 values were calculated from thecalibration curve and associated regression equation, shown in FIG. 10A.As evident from the data plot in FIG. 10B, GRI therapy alone improvedPbtO2 significantly as compared to vehicle-treated RIC-mock operatedgroup. RIC therapy alone did not improve PbtO2 in diabetic stroke ascompared to vehicle-treated stroke group; however, in combinationtherapy group, a prior treatment with GRI followed by RIC therapy showedenhanced efficacy of both, GRI and RIC-therapies, in improving benefitsof reperfusion to increase PbtO2.

Mice were immediately sacrificed after PbtO2 measurement and braintissue samples were analyzed for inflammatory gene expression (N=6/gp).RT-PCR primers shown in Table 1 were used to measure changes inexpression of iNOS (FIG. 11A), iCAM-1 (FIG. 11B), IL-1β (FIG. 11C) andIL-6 (FIG. 11D). The result for each inflammatory gene was normalized toexpression of β-actin and expressed as a fold change.

TABLE 1 Nucleotide Sequences of Mouse Primers used for RT-PCR. GenbankGene Accession No. Primer sequence SEQ ID NO iNOS NM_010927.3GAT GTG CTG CCT CTG GTC TT SEQ ID NO: 1 TT GGG ATG CTC CAT GGT CACSEQ ID NO: 2 iCAM-1 NM_010493 ACG CAG AGG ACC TTA ACA GTC TACSEQ ID NO: 3 GCT TCA CAC TTC ACA GTT ACT TGG SEQ ID NO: 4 IL-1βNM_010554.4 GCA CCT TAC ACC TAC CAG AGT SEQ ID NO: 5AAA CTT CTG CCT GAC GAG CTT SEQ ID NO: 6 IL-6 NM_031168.1TAG TCC TTC CTA CCC CAA TTT CC SEQ ID NO: 7 TTG GTC CTT AGC CAC TCC TTCSEQ ID NO: 8 β-actin NM_007393 TGA CAG ACT ACC TCA TGA AGA TCCSEQ ID NO: 9 ACA TAG CAC AGC TTC TCT TTG ATG SEQ ID NO: 10

GRI therapy alone showed a trend only to downregulate most inflammatorygenes and were not significantly different from the vehicle-treatedstroke group. Surprisingly, RIC therapy alone enhanced inflammatoryresponses in stroke-induced diabetic mice; however, in the combinationGRI+RIC therapy group, prior treatment with GRI significantlydownregulated and prevented RIC-induced inflammatory responses indiabetic stroke mice as compared to the RIC therapy alone group.

Example 5

GRI Enhances Neuroprotective Efficacy of RIC therapy intMCAo-STZ-induced Diabetic Mice.

Diabetic adult male mice (as described in Example 4) were subjected totMCAo (N=10/gp) with or without GRI and/or RIC-therapies and outcomemeasures were performed at 24 h post-stroke. A 2×2 factorial ANOVA wasused to analyze results, data are expressed as Mean±SD, indicating Meanswith different letters are significantly different (P<0.05).

Looking at the results for mice receiving a vehicle treatment instead ofGRI and/or RIC, treatments including any sham component (vehicle-mockoperated control, GRI-treatment singly and RIC therapy alone) did notimprove any of the neurological outcomes of mean NDS, % infarct volume,or % swelling, shown in FIGS. 12A, 12B, and 12C, respectively. However,when low-dose GRI was combined with RIC therapy, all outcome measureswere significantly improved in combination therapy group as compared toall other 3 groups. Representative TTC-stained fresh brain tissueimages, shown in FIG. 12D, further supports the finding that GRI incombination with the NO-enhancing RIC therapy increases neuroprotectionin diabetic stroke.

Example 6

GRI Therapy Protects against TE Stroke Injury and Enhances Benefits ofLate-IVT Therapy. In this example, the TE-clot eMCAo model is used todemonstrate a method of the invention. Adult male WT-mice (16±2-weeksold; N=20/gp) were subjected to eMCAo using a partially humanizedTE-clot (9±1-mm). Mice were randomized to 4-groups: 2 GRI (GRI vs. Veh)by 2 IVT (IVT vs. Veh). GRI therapy (2.5 mg/kgbwt) or equal volume ofvehicle was IV-infused at 1 h post-eMCAo. The same dose of GRI therapyor equal volume of vehicle was also repeated and IP-injected after IVTor IVT-veh at 6 h and 24 h post-eMCAo. Late IVT-therapy (tPA as Altepaseformulation; 10 mg/kg in 250 uL) or equal volume of vehicle was infusedat 5 h post-eMCAo as 10% of volume IV bolus and remaining over a periodof 20 min. A 2 GRI (GRI vs. Veh) by 2 IVT (IVT vs. Veh) ANOVA was usedto analyze results, as appropriate. All the data are expressed asmean±SD, and pairs of means indicated with different letters aresignificantly different (P<0.05).

For stroke injury/edema volume measurement, 6 surviving mice from eachgroup were randomly selected for T2-Weighted MRI at 24 h post-eMCAo. Asevident from the volume quantification shown in FIG. 13A, early GRItherapy alone after stroke prevented the edema progression significantlyas compared to Veh-treated group and also in comparison to late-IVTtherapy. The representative coronal slices from the MCA region shown inFIG. 13B further support the quantitative finding shown in FIG. 13A.Moreover, as anticipated, late-IVT therapy did not prevent the edemaprogression/stroke injury significantly as compared to Veh-treatedgroup. However, treatment with GRI therapy after eMCAo and prior totPA-injection enhanced the benefits of late IVT and prevented edemaprogression significantly as compared to both, Veh- and IVT-treatedgroups.

Neurological deficit scoring (NDS) based on a modified Bederson Scalewas performed for all surviving mice at 96 h post-eMCAo. As evident fromthe mean NDS values shown in FIG. 14A, early GRI therapy alone afterstroke reduced the later neurological deficit significantly as comparedto both Veh- and late-IVT-treated groups. Late-IVT therapy failed toimprove the neurological score; however, GRI therapy in combination withlate-IVT therapy significantly improved the neurological outcomes at 96h post-stroke. Kalan-Meier survival curve for all 4 groups followed for2 weeks. As evident from the curve shown in FIG. 14B, both groupswithout a GRI treatment showed higher mortality within the initial 5days (acute/sub-acute phase) after stroke with a prominent trend inmortality in late-IVT treated group. A higher trend in mortality inlate-IVT treated group is likely attributed to frequent hemorrhagictransformation, as indicated (asterisk) in the representative coronalsection for IVT-group in FIG. 14B.

On day 13 & 14, all surviving mice were tested for long-term functionaloutcomes such as the recovery of muscular strength (hanging wire test),shown in FIG. 15A, and protection of learning/memory function (NORtest), shown in FIG. 15B. In agreement with the benefits of acuteprotection, GRI therapy alone and in combination with late-IVT therapysignificantly improved and protected the long-term motor andlearning/memory functions, respectively, compared to both Veh- andlate-IVT-treated groups. As such, late-IVT beyond 4.5 h post-strokeremained ineffective in improving long-term functional outcomes ascompared to Veh-treated mice.

At day-15 post-eMCAo, all mice were sacrificed after behavioral outcometests and brains were harvested. Q-ball MRI (DTI, T1-Intensity and DWIsequences) of randomly assigned representatives of each group wasperformed ex vivo using fixed brain samples. Results are shown inrepresentative series of sections for each treatment group in FIG. 16.As evident from the appearance of the injured hemisphere (strokedipsilateral side), both Veh- and late-IVT-treated groups demonstratedhigher cerebral atrophy, larger ventricle size and greater loss in whitematter (WM; corpus callosum) integrity. In contrast, GRI-treated groupswith and without late-IVT showed preservation of WM-integrity, reducedcerebral atrophy and decrease in ventricle size enlargement.

The foregoing examples of the invention demonstrate the following novelfindings:

-   -   1. The changes in expression and activity GSNOR have never been        investigated and reported in stroke models, particularly in the        most clinically relevant partially humanized TE stroke model.        For the first time, these examples demonstrate that GSNOR        expression/activity is increased within 3-6 hours after stroke.    -   2. A dose-escalation study for GRI therapy with validation in        different stroke models, different ages and sexes, and various        dynamics of reperfusion, has never been performed. For the first        time, the Examples of the invention demonstrate differential        effects of GRI therapy in various clinically relevant settings,        such as different dynamics of reperfusion (permanent occlusion,        partial reperfusion, complete reperfusion simulating IVT and        EVT), aged vs young rodents, and male vs female.    -   3. The GRI therapy has not previously been tested in any kind of        comorbid (diabetic, hypertensive) stroke models with or without        reperfusion. For the first time, the Examples of the invention        demonstrate therapeutic benefits of GRI therapy in comorbid        strokes models in both, reperfused and non-reperfused stroke        models.    -   4. RIC therapy is a modulator of endogenous NO level and a        recent outcome report informs that the overall benefit of RIC        therapy was neutral, i.e., without any benefits. Of note, a        large population of stroke patients have comorbidities (Pico et        al. Int J Stroke. 2016; 11(8):938-943 (see also,        pubmed.ncbi.nlm.nih.gov/31500849,        pubmed.ncbi.nlm.nih.gov/31621833,        pubmed.ncbi.nlm.nih.gov/27412192, and        pubmed.ncbi.nlm.nih.gov/29748420)). The Examples of the        invention provide the first demonstration that RIC therapy alone        is not protective in comorbid stroke models. These Examples also        demonstrate for the first time that in comorbid        (diabetic/hypertensive) strokes, performing GRI therapy prior to        RIC enhances the benefits of RIC, turning it into an effective        therapy in ischemic stroke with comorbidity.    -   5. Since it is important to demonstrate the safety of GRI        therapy with the FDA-approved IVT, The Examples also demonstrate        for the first time that the GRI therapy in combination with IVT        therapy in the most clinically relevant partially humanized TE        stroke model enhances the benefits of IVT and extends its safe        window of thrombolysis.    -   6. Using state-of-art live animal imaging (MRI and LASCA) and in        vivo brain tissue oxygenation recording techniques, the Examples        of the invention demonstrate for the first time that GRI therapy        improves microcirculation and brain tissue oxygenation.

In summary, the methods of the invention demonstrate the efficacy of GRItherapy as an adjunct therapy administered with RIC therapy in differentmodels of stroke, particularly comorbid stroke, and under variousclinically relevant conditions. GRI therapy showed a dose dependentdifferential effect, enhanced neurovascular protection and behavioraloutcomes, and remain safe and beneficial with FDA-approved therapies andtherapies under trial such as RIC.

While the invention has been described in terms of its several exemplaryembodiments, those skilled in the art will recognize that the inventioncan be practiced with modification within the spirit and scope of theappended claims. Accordingly, the present invention should not belimited to the embodiments as described above but should further includeall modifications and equivalents thereof within the spirit and scope ofthe description provided herein.

ACKNOWLEDGMENT

“This project was funded by the National Plan for Science, Technologyand Innovation (MAARIFAH)—King Abdulaziz City for Science andTechnology—the Kingdom of Saudi Arabia—award number (14-MED2597). Theauthors also, acknowledge with thanks Science and Technology Unit, KingAbdulaziz University for technical support”.

We claim:
 1. A method of treating a hypertensive subject who issuffering a blockage of brain microcirculatory flow caused or suspectedto be caused by a thromboembolic stroke, comprising the steps ofperforming a remote ischemic conditioning (RIC) therapy to induceproduction of endogenous nitric oxide, and while performing the RICtherapy, administering a therapeutically effective amount of anS-nitrosoglutathione reductase (GSNOR) inhibitor (GRI therapy) selectedfrom the group consisting of N6022, cavosonstat, N91115 and N6338,wherein the therapeutically effective amount of GSNOR inhibitor issufficient to preserve and enhance the activity of the endogenous nitricoxide and inhibit brain tissue injury; and administering thereafter andintravenous tissue plasminogen activator therapy (IVT) for thrombolysis,wherein the therapeutically effective amount of the IVT is sufficient todissolve a thrombus or a blood clot from an artery, wherein the RICtherapy, the GRI therapy and the IVT reduce the risk of reperfusioninjury in the hypertensive subject.
 2. The method of claim 1, whereinthe GSNOR inhibitor is N6022.
 3. The method of claim 1, wherein thehypertensive subject also suffers from diabetes or type II diabetes.